Myocardial infarction and heart failure represent the main cause of death in industrialized countries. The loss of terminally differentiated cardiac myocytes which is associated with these pathologies accounts for a decrease in myocardial function which can lead to total organ failure or trigger compensatory mechanisms like hypertrophy of the remaining myocardium, activation of neurohumoral systems, and autokrine/parakrine stimulation by various growth factors/cytokines.
It has been shown that endogenous regenerative mechanisms do not suffice to compensate for cardiac myocyte death after myocardial infarction. Modern pharmacotherapy can delay, but not reverse the natural course of the disease. Thus, exogenous regenerative strategies including cardiac implantation or coronary transfusion of cells, activation of endogenous cardiac regeneration by pharmacological means, or implantation of performed, engineered cardiac tissues have gained increasing attention (Reinlib, L. & Field, L., Circulation 101, E182-7 (2000); Zimmermann, W. H. & Eschenhagen, T., Heart Fail Rev 8, 259-69 (2003); Olson, E. N., Nat Med 10, 467-74 (2004); Orlic, D. et al., Proc Natl Acad Sci USA 98, 10344-9 (2001)).
So far, cells of various origins and developmental stages have been grafted into healthy and diseased hearts, including immature cardiac myocytes (Reinecke, H., Zhang, M., Bartosek, T. & Murry, C. E.; Circulation 100, 193-202 (1999); Muller-Ehmsen, J. et al., Circulation 105, 1720-6 (2002); Li, R. K. et al., Circulation 96, 11-179-86; discussion 186-7 (1997)), skeletal myoblasts (Taylor, D. A. et al., Nat Med 4, 929-33 (1998); Menasche, P. et al., Lancet 357, 279-80 (2001)), fibroblasts (Etzion, S. et al., Circulation 106, 1125-30 (2002); Sakai, T. et al., J Thorac Cardiovasc Surg 118, 715-24 (1999)), endothelial cells (Condorelli, G. et al., Proc Natl Acad Sci USA 98, 10733-8 (2001)), embryonic stem cell-derived cardiac myocytes (Soonpaa, M. H., Koh, G. Y., Klug, M. G. & Field, L. J., Science 264, 98-101 (1994)), and a number of potential cardiac progenitors from peripheral blood (Assmus, B. et al., Circulation 106, 3009-17 (2002)) or bone marrow (Orlic, D. et al., Nature 410, 701-5 (2001); Stamm, C. et al., Lancet 361, 45-6 (2003); Strauer, B. E. et al., Circulation 106, 1913-8 (2002)). Even though the evidence for the formation of true cardiac muscle tissue, electrically coupled to the host myocardium is sparse, most studies reported procedure-induced improvement of contractile function after cryo-injury or myocardial infarction.
A different concept in cardiac regeneration is to graft heart muscle produced ex vivo by tissue engineering. Tissue engineering aims at generating functional 3-dimensional tissues outside of the body that can by tailored in size, shape and function according to the respective needs before implanting them into the body. In contrast to cell implantation in preexisting structures, this approach may allow for complete replacement of diseased myocardium or reconstitution of missing cardiac structures in individuals with cardiac malformations.
Despite its potential, cardiac tissue engineering is still in its infancy for several reasons: (i) Postnatal cardiac myocytes do not or not sufficiently replicate. Given the high absolute numbers of cells needed for cardiac regeneration (Gepstein, L., Circ Res 91, 866-76 (2002)), utilisation of primary cardiac myocytes will not be feasible. (ii) To repair myocardial infarctions or correct heart defects, heart tissue patches need to be engineered at a size and with contractile features that have a realistic chance to lend significant support to failing hearts. Size as well as function and in vivo survival depend crucially on metabolic supply. Thus, vascularization is likely to be a prerequisite for successful creation and grafting of large cardiac tissue patches. (iii) The heart not only needs systolic strength, but also depends on unimpeded diastolic relaxation. Consequently, engineered heart muscle must exhibit a large compliance, a feature often neglected in classical, scaffold-based tissue engineering approaches. (iv) Finally, any successful tissue engineering concept will depend on structural and electrical integration of implanted tissue into the host myocardium. Recent progress in stem cell research has opened new perspectives for cell sourcing, e.g. from embryonic (Kehat, I. et al., J Clin Invest 108, 407-14 (2001)) or adult stem cells (Beltrami, A. P. et al., Cell 114, 763-76 (2003); Messina, E. et al., Circ Res (2004)).
Several publications describe the generation of engineered heart tissue in different geometrical sizes and shapes (Zimmermann, W. H. et al., Circ Res 90, 223-30 (2002); Fink, C. et al., Faseb J 14, 669-79 (2000); see also published International Application WO 01/55297). It was shown that engineered heart tissue can be reconstituted by mixing heart cells from rat (including cardiac myocytes, fibroblasts, smooth muscle cells, endothelial cells, macrophages and other cells of leukocytic origin, etc.) with collagen type I, matrigel and serum-containing culture medium (the complete mixture is referred to as reconstitution mix). Specifically, ring-shaped casting molds were used to form ring-shaped EHTs. The engineered tissue rings resemble intact heart tissues in terms of force-frequency behaviour, force-length relationship (Frank-Starling mechanism) and response to extracellular calcium (Eschenhagen T, et al., Faseb J. 11, 683-94 (1997); Zimmermann W H et al., Biotechnol Bioeng. 68, 106-14 (2000). These data support the conclusion of true heart tissue-development in vitro from a functional point of view. However, differences exist as well. For example, absolute contractile forces of EHTs remain lower than in the intact heart. In the literature, maximal twitch tensions of about 1-2 mN/mm2 are reported in artificial heart muscle (overview in Zimmermann W H, et al., Heart Fail Rev. 8, 259-69 (2003)). In comparison, papillary muscles from rat develop 4-10 mN/mm2. The difference of EHT force generation as compared to mature heart muscles most likely reflects both a quantitative and a qualitative aspect, as for example a lower fractional occupancy of the EHT tissues by cardiac myocytes and the lower degree of sarcomere development. In fact, single adult cardiac myocytes develop up to 56 mN/mm2 These ideal forces are unlikely to be reached in intact muscle preparations due to the lack of oxygen and metabolite supply in the absence of blood perfusion.
It has been demonstrated that a simple scale-up of the preparation approach, e.g. by use of larger casting molds than those described in International Application WO 01/55297 cannot solve the problem, since size of the engineered tissue constructs appears to be limited by maximum diffusion distances for nutrients and oxygen. Indeed, none of the various tissue engineering approaches developed today generate cardiac tissue-like, contracting constructs of a thickness of more than 0.8 mm (Zimmermann, W. H. et al., Circ Res 90, 223-30 (2002)). Yet, so far developed artificial heart tissues do not represent homogeneous myocardium, but consists of a large fraction of cell-free matrix and interconnected cardiac muscle strands that do not exceed 20-100 pm in thickness. Interconnected muscle strands can be observed and evaluated by confocal laser scanning microscopy e.g. after actin or actinin staining as outlined in Zimmermann et al. (Circ Res 90, 223-30 (2002)).
Therefore, a need exists for the provision of improved heart tissue grafts of larger size which overcome the above shortcomings. It has been an object of the present invention to provide heart tissue grafts having a size and contractile strength which allows for the effective support of failing hearts in a mammal, such as a human. The present invention provides for a solution of this problem and offers other related benefits as well.
It has surprisingly been found in course of the invention that large threedimensional tissue grafts which consist of a well-organized and highly differentiated cardiac muscle syncytium can be produced by fusing two or more engineered heart tissue rings with each other. Fusing, as used herein, means that single EHT units or stripes grow together at a region where sustained physical contact is established to form a contiguous cell assembly. The assembly represents a structurally, electrically and functionally syncytium that may be finally used as a cardiac tissue graft in vivo. Such graft constructs are designated “multiring engineered heart tissue construct” herein. Specifically, it has been found that several engineered heart tissue rings or comparable geometric forms can be woven together, thus forming a network in which each individual construct remains accessible for unlimited diffusion and exchange of nutrients. As a consequence, these constructs do not exhibit the typical size limitations known from the prior art. Multiring engineered heart tissue constructs of the invention can be produced by using a plurality of distinct engineered heart tissue rings as those described in the prior art. The rings can be fused in any suitable manner to provide “chain-mail”-like constructs. By this technique, the form of the multiring constructs can be adjusted dependent on the specific therapeutic approach. For example, large multiring netlike patches can be generated for replacing dysfunctional areas of the heart of a mammal. Alternatively, bag-like forms may be generated which surround the complete organ. The size of the multiring construct patches can be varied dependent on the size of the area to be treated. The multiring engineered heart tissue construct of the invention can be comprised of force-generating engineered heart tissue rings derived from any mammal. In view of its intended use in the field of tissue augmentation, it is preferred that the engineered heart tissue rings are derived from human cells.
Thus, according to a first aspect of the invention, an ex vivo method for the preparation of a multiring engineered heart tissue construct suitable for use in cardiac tissue augmentation and/or replacement therapy is provided, which method comprises the steps of                a) providing force-generating engineered heart tissue rings;        b) placing into contact at least two force-generating engineered heart tissue rings so that each force-generating engineered heart tissue ring has one or more contact points to an adjacent force-generating engineered heart tissue ring; and        c) culturing the force-generating engineered heart tissue rings under conditions which allow fusion of the at least two force-generating engineered heart tissue rings at the one or more contact points to form a multiring heart tissue construct.        
As used herein, the term “multiring engineered tissue construct” means an ex vivo produced tissue construct comprised of at least two, and preferably more than two distinct force-generating engineered heart tissue rings, which may have been produced by tissue engineering techniques, such as those described in Zimmermann, et al., Circ Res 90, 223-30 (2002) and International Application WO 01/55297. The term “tissue” refers to a group of similar cells united to perform a specific function or a grouping of cells that are similarly characterized by their structure and function. The constructs of the invention consist of at least two distinct engineered heart tissue rings. In this context, the expression “multiring” is meant to emphasize that the fused construct comprises a plurality of loop structures originating from the essentially circular shape of the force-generating engineered heart tissue rings that serve as a basis material for the construct of the invention. Such loop structures have been proven very useful for the handling of the tissue construct, for example when surgically fixing the construct to the recipient organ or tissue. Additionally, the term multiring is also intended to comprise embodiments in which tissue constructs are formed which consist of tube- or stripe-like structures. The former might be obtained by establishing side-to-side connections between two or more rings (FIG. 8B). The latter might be obtained by forming a ring structure and subsequently cut the latter in two or more pieces for further use. An example of such tissue construct is provided in FIG. 8C.
The multiring construct according to the present invention comprises force-generating engineered heart tissue rings. The production of such contractile engineered heart tissue in circular/annular shape has been described in detail in the prior art, for example in published International Application WO 01/55297 and in Zimmermann et al., 2000, Biotechnology and Bioengineering Vol. 68, pages 106 to 114. Cardiac myocytes such as those obtained from tissue samples of neonatal hearts of different mammals may be used for preparing the EHT rings, as described in Zimmermann, et al., Circ Res 90, 223-30 (2002) and International application WO 01/55297. As can be taken from these publications, engineered heart tissue rings can be obtained by culturing cardiac myocytes in ring-shaped molding devices. It has been shown that mixed populations consisting of almost identical portions of cardiac myocytes and non-cardiac myocytes result in EHTs with increased contractile properties compared to selected cardiac myocytes (Zimmermann et al. (2003), Heart Failure Rev., 8, 259-269). Fetal or neonatal cardiac myocytes from mammals, such as rat, mouse or primates are a suitable source for obtaining EHT rings. In contrast to differentiated cardiac myocytes, these cells still have the capability to divide as well as to redifferentiate after dedifferentiation. Briefly, a solution of a scaffold substance (such as collagen type I) is mixed with culture medium (final concentration in the mixture: 1×DMEM; 2% chicken embryo extract; 10% horse serum; 100 jig/ml Streptomycin; 100 U/ml penicillin). The pH of the mixture is adjusted to physiologic values . . . 7.4 with 0.1 N NaOH. Engelbreth-Holm-Swarm tumor exudate (also known as “Matrigel”) is added to give a final concentration of 5-15%. This mixture is added to a cell suspension of fetal or neonatal cells (for example, 2.5×106 cells/EHT). Alternatively, serum-free media may be used. A serum-free medium suitable for culturing the cardiac myocytes is exemplified in FIG. 12. The person skilled in the art will appreciate that several modifications as to the components of the medium can be performed. Moreover, Matrigel may be substituted against insulin and triiodothyronine, as described in more detail below.
Alternatively, mammalian cardiac myocyte progenitor cells, such as pluripotent stem cells, adult or embryonic, provide a source for preparing the EHT rings. Preferably, the EHT rings to be used in the method according to the invention are derived from human cells. Human engineered heart tissue rings may be obtained by use of circular-shaped casting molds. In this case, cardiac myocyte progenitor cells, such as stem cells, more preferably pluripotent embryonic stem cells, may be used for preparing the force-generating engineered heart tissue rings. However, also other types of stem cells, such as pluripotent adult stem cells may be used. The preparation of contractile EHT rings derived from human stem cells is particularly described in example 6 of the present invention.
The force-generating engineered heart tissue rings produced from human or non-human mammalian cells comprise cardiac myocytes. However, they are usually not composed purely of cardiac myocytes but comprise almost all cells species that are normally found in the heart including cardiac myocytes, fibroblasts, smooth muscle cells, endothelial cells, macrophages and other cells of leukocytotic origin. Typically, the presence of cardiac cells within a given EHT ring can be confirmed by positive staining with anti-cardiac myosin heavy chain, anti-α-actinin, anti-desmin and/or anti-cardiac troponin I antibodies (see Kehat I. et al, supra). If cells are derived from human cells, this can readily be confirmed by well known methods, such as PCR analysis based on typically conserved regions of the genome.
The term “ring” as used in the context of the present invention is meant in a broad sense to comprise also geometric forms which do not represent a perfect geometric ring. For example, it may also comprise other forms which consists of annularly closed tissue tube structures. It has been found that a circular geometry of the EHT is ideal for several technical and biological reasons. For example, circular heart tissue structures allow large scale production with minimal handling. Most importantly, circular engineered heart tissue structures can be easily miniaturized for high-throughput screening and they exhibit better tissue formation than the non-circular designs, since the circular form causes homogeneous force distribution throughout the tissues. Furthermore, nutrient distribution is facilitated in circular-shaped tissues. Also not being particularly restricted to a specific limited size, it has been shown that engineered heart tissue rings having an outer diameter of 8-12 mm have particularly preferred characteristics. The inner diameter of these rings should be in the range of 6-10 mm. Particularly preferred are EHT rings with an outer diameter of about 10 and an inner diameter of about 8 mm (see International Application WO 01/55297).
As used herein, the term “force-generating” means that the engineered heart tissue rings are able to actively contract against a given mechanical load. The intrinsic property of heart cells to form contractile aggregates has been known in the field for years. Several publication reported that contractile properties are maintained when threedimensional EHTs, such as EHTs of circular shape, are constructed. Contraction of the tissue constructs are triggered by addition of Ca2+ ions or isoprenaline to the culture medium. Zimmermann, W. H. et al., Circulation 106, I 151-7 (2002), reported determination of contractile functions of engineered tissue rings by isometric force measurements in organ baths before implantation (see FIG. 2a of the above publication). Maximal twitch tension (TT) values of about 0.4 mN to 0.7 mN were measured.
According to a first step of the method for the preparation of the multiring engineered heart tissue construct of the invention, distinct force-generating engineered heart tissue rings are provided. These rings are, for example, obtainable by culturing suitable cells in ring-shaped molding devices. The preparation of such rings from mammalian cell sources is described in the art, for example, in Zimmermann, et al., Circ Res 90, 223-30 (2002), in International Application WO 01/55297 and in Example 1. Using the same casting molds, engineered heart tissue rings may also be prepared from human stem cells, for example, as described in Example 6.
Subsequently, at least two force-generating engineered heart tissue rings are positioned into contact, so that each of these force-generating engineered heart tissue rings has one or more contact points with at least one further EHT ring in its vicinity. This means, each of the engineered heart tissue rings used for preparing the multiring heart muscle construct is tangent to at least a further engineered heart tissue ring. Preferably, the distinct EHT rings are held via suspension points of a holding device. According to a very simple embodiment of the invention, several force-generating engineered heart tissue rings as defined herein are stacked, i.e. laid upon each other to form a stack with a plurality of loops extending in different directions (see for example FIG. 8A). It is particularly preferred that the force-generating EHT rings are placed into contact by stacking of the distinct rings to form a central region in which the force-generating engineered heart tissue rings overlap with each other as shown in FIG. 8A). According to an alternative embodiment several rings are placed into contact as shown in FIG. 8B. Apart from that, any other random distribution of the tissue rings in which each ring is in contact with at least another one are possible. It has been found in course of the invention, that the tissue rings which have been positioned into contact in this manner fuse and form a complex in-unison contracting construct which is synchronized with respect to its contracting activity. This finding allows for the generation of large tissue-like structures by fusing distinct contractile EHT rings (or perfused multiring structures comprising several of theses EHT rings).
In a further step, the force-generating engineered heart tissue rings, which are in contact with each other, are cultured under conditions which allow the fusion of the contacted ring structures to form an artificial heart muscle construct. For this purpose, the engineered heart tissue rings are normally incubated in culture dishes, wherein the rings are submersed in appropriate culture medium. If a holding device is used for culturing, the device should be designed to allow the tissue rings to stay in continuous contact with the culture medium. The choice of the culture medium is not critical. Conventional media for culturing cardiac myocytes may be used. Such media are known in the art and described, for example, in Zimmermann, W. H. et al., Circulation 106, I 151-7 (2002) and published International Application WO 01/55297. These media may further be modified, i.e. by substituting Matrigel against a mixture of Insulin (10 pg/ml) and triiodothyronine (1 nM). It was found that the addition of insulin and triiodothyronine allowed for the generation of strongly contracting EHTs. Simultaneous addition of both factors for only 24 h at the beginning of the EHT culture was sufficient. By using insulin and triiodothyronine during EHT construction, improved EHT contractility was achieved. These effects did not stem from enhanced overall EHT cell number, since no difference in the DNA content and no apparent structural differences were observed. Rather, the effects may be the result of enhanced protein content or improved survival of cardiomyocytes leading to a higher cardiomyocyte fraction without an apparent change of the overall cell number (standard EHTs on culture day 12 contain roughly 100.000 cardiomyocytes and a total cell number of 600.000; a fractional increase in cardiomyocyte number may have significant effects on force of contraction, may lead to an increase in overall “cardiomyocyte” protein [e.g. sarcomeric actin] but may not lead to a measurable [by total DNA quantification and cell counting after enzymatic digestion of EHTs) increase in total cell number in EHTs treated with insulin and triiodothyronine. Furthermore, the media as used herein for culturing stem cells in example 6 can be used as well.
According to a more preferred embodiment of the invention, the same media which has been used for casting the EHT rings are used during incubation of the force-generating engineered heart tissue rings for fusion (see examples 1 and 6 for neonatal cardiomyocyte and stem cells, respectively). Suitable culturing conditions are described in the art and comprise a physiological temperature range of 30-40° C., preferably 3638° C., and more preferably 37° C. The percentage of O2 in the ambient air should range from 21-80%, preferably 70%, 60% 50%, and more preferably 40%. Culturing may be performed for 3 to 50 days, preferably 10-30 days, and more preferably 15-20 days.
According to preferred embodiment of the present invention a plurality of distinct force-generating engineered heart tissue rings are placed into contact to form the fused multiring EHT construct. Preferably, five or more tissue are placed into contact, for example six, seven, eight, nine, ten, eleven, twelve or more. More preferably, more than 15, 20, 30, 40, 50, or even up to 100 force-generating engineered heart tissue rings are placed into contact to form the fused multiring EHT construct. It has to be understood that larger multiring EHT constructs can also be formed in several steps, for example by forming several constructs of 5-10 EHT rings in a first step and fusing these different constructs in a subsequent step. In any case, care should be taken that the resulting multiring EHT construct is cultured for a further period of time to allow synchronization of contractile activities.
According to preferred embodiment of the present invention, the force-generating engineered heart tissue rings used for preparing the multiring heart muscle construct of the invention are subjected to tensile stress prior to or simultaneously with placing into contact the at least two force-generating engineered heart tissue rings. For example, when using a device of the invention as described in more detail below, placing into contact of the EHT rings and subjecting to tensile stress can be performed in one step. Subjecting to tensile stress can for example be performed by actively stretching the tissue rings, for instance by use of a mechanical stretching device like the one described in Zimmermann, W. H. et al., Circulation Res 90, 223-30 (2002). Such device can provide for a “static” load, i.e. a permanent load produced by expanding the ring by 3 to 20%, preferably 10% of its original length (original length determined in the absence of any load) as described in WO 01/55297. Alternatively, a “phasic” load can be applied by periodically expanding the ring by 3 to 20%, preferably 10% of its original length, for example at a frequency of 0.1-10 Hz, preferably 1-5 Hz. Such stretching can be regarded as a static or phasic manner, respectively.
In several publications, it was demonstrated that contractile properties of artificially synthesized heart tissue rings can be improved by mechanically stretching the rings after there casting (see Eschenhagen et al., 2002; Fink et al., 2000; Zimmermann et al., 2002b). It has been demonstrated that mechanical load improves both orientation and differentiation of muscle cells in EHT rings. Tensile stress can be applied for a period of time ranging from several hours to more than 40 days. Preferably, tensile stress is applied for 5-9, more preferably 7 days. During imposing tensile stress, the EHT rings are cultured under the conditions recited above.
According to a preferred embodiment of the invention, tensile stress is applied by a static, phasic or auxotonic manner or a combination thereof. According to a particularly preferred embodiment of the invention, tensile stress is applied by a auxotonic manner. An “auxotonic manner”, as used herein, means that the engineered heart tissue rings are kept under conditions so that they have to contract against a defined force and are reexpanded during relaxation of the EHT ring by another force or, preferably, the same force. For example, if the tissue ring is held under tension by using a spring, this spring will provide a defined force against which the EHT must contract. Upon relaxation of the EHT ring, the spring will expand the EHT ring again. It has been found that culturing the EHT rings in this manner leads to a considerable increase of contractile properties compared to phasic and static processes (see FIG. 1B). Therefore, both the single engineered heart tissue rings as well as the artificial multiring heart muscle construct consisting of the distinct heart tissue rings show improved maximal active forces during contraction. Under auxotonic load, the EHT rings have to perform contraction under conditions which resemble the systole and diastole phase of the heart cycle. In other words, the engineered heart tissue rings are held under conditions which corresponds to the native environment of active heart tissue.
Auxotonic tensile stress can be applied, for example, by elastically suspending each of the at least two force-generating engineered heart tissue rings between at least two associated suspension means wherein at least one of said associated suspension means is resiliently biased (i.e. mounted to be movable against a bias force) so that the individual force-generating EHT rings are able to contract against the bias force provided by the suspension means and are reexpanded during relaxation of the EHT ring. Preferably each of the force-generating engineered heart tissue rings is suspended between two associated suspension means. Preferably, the two suspension means can be variably adjusted to be used with rings of different diameters. The suspension means may be part of a device as explained in detail below. Such device comprises a pair of suspension means for each EHT ring. According to a further embodiment, all of the associated suspension means are resiliently biased.
Preferably, the at least one suspension means can be adjusted to vary the tensile stress. This means, the load applied to the EHT rings can be easily adjusted to a specific strength by modulating the tension provided by the resilient means. For example, springs can be used as resilient means comprising spring coils which can be adjusted in order to modulate the resilient tension of the spring.
As already mentioned, “auxotonic” tensile stress can be applied simultaneously with placing into contact of the at least two force-generating engineered heart tissue rings. For example, the different EHT rings can be suspended in a device as explained in detail below so that the tissue rings are held in a position that provides for a plurality of contact points between the different heart tissue rings. It has been found that the fusion of the different rings at their contact points occurs also during the different rings are subjected to the tensile stress. Surprisingly, subjecting the heart tissue rings to tensile stress in terms of auxotonic (or phasic or static) load obviously does not hamper formation of a fused multiring construct. Therefore, the culturing which allows fusion of the at least two engineered heart tissue rings to form an artificial heart muscle construct can be performed while the heart tissue rings are held under tensile stress.
According to a further aspect, the invention refers to multiring EHT constructs which comprise at least two force-generating engineered heart tissue rings fused with each other to form an interconnected tissue construct. The multiring engineered heart tissue construct is obtainable according to a method described above. As already explained, the multiring constructs can comprise more than 5 EHT rings, for example 15, 20, 30, 40, 50, or even up to 100. Preferably, each of the EHT rings has an outer diameter of 8-12 mm.
Preferably, the multiring EHT constructs of the invention are comprised of fused engineered heart tissue rings which are stacked to form a central region in which the force-generating engineered heart tissue rings overlap with each other. A construct, which results from stacking of five different tissue rings is depicted in FIG. 8A. Further examples of multiring EHT construct structures are provided in FIG. 8B and FIG. 8C. FIG. 8B shows a plurality of ring structures which are suspended by a flexible lateral suspension device wherein the distinct ring structures are in contact with each other in the region of contact with the suspension means. It has to be understood by the person ordinary skilled in the art that these examples of ring assemblies are not limiting. A large number of further geometric structures are conceivable for example bag- or net-shaped structures or different forms of stackings.
The multiring heart muscle constructs provided by the present invention show properties which makes them particularly useful for tissue grafts in heart tissue augmentation and/or replacement therapy. In animal models, it has been shown that multiring heart muscle constructs generated from neonatal myocard cells of rats were suitable to provide functional tissue grafts which could be successfully transplanted to infarcted rat hearts and support dysfunctional tissue damaged after myocard infarct (see examples). The multiring engineered heart tissue construct comprise cardiac myocytes, i.e. cells comprising one or more of the following proteins: cardiac myosin heavy chain, α-actinin, desmin and/orcardiac troponin I. It is clear that tissue grafts which are intended to be used in human therapy should be derived from cells of human origin. Therefore, according to preferred embodiment of the present invention, the multiring heart muscle constructs are comprised of force-generating engineered heart tissue rings derived from human cells. The human origin of the EHT can be confirmed by a plurality of different methods known in the art, for example by amplification of species-specific DNA sequences and/or the antibody-based detection of species-specific antigens, such as polypeptides. The preparation of the engineered heart tissue rings starting from human stem cells is exemplified in detail in example 6 of the present invention.
It is preferred that the multiring EHT construct of the invention has a overall twitch tension of more than 2.5 mN, more preferably more than_3 mN, for example 3.5 mN, 4 mN, 4.5 mN, 5 mN, 5.5 mN, 6 mN, 10 mN, 15 mN or more. The capability of the multiring construct to contract can be determined in accordance with methods as described in the prior art, for example, by monitoring the constructs in standard organ baths (see Zimmermann et al., Biotech. Bioeng., 68, (2000)).
According to a further aspect, the invention relates to the use of the multiring EHT construct of the invention for drug target validation and drug development. According to a preferred embodiment, the multiring EHT construct is used in drug screening or target validation assays. In this context, it is preferred that the multiring EHT construct used is derived from a mammal, such as a human. For example, the multiring construct can be used to analyze the capability of candidate drugs to interfere with the physiological function of the native cardiac tissue of a mammal. For this, the capability of said candidate drug to enhance or reduce contractile functions of the multiring EHT construct is determined. Moreover, the multiring EHT construct may be used in determining the influence of the activity of certain genes on the physiological function of the native cardiac tissue of a mammal (referred to herein as target validation). For this purpose, genes of the cells within the distinct EHT rings (or cells which are used for preparing the same) may be knocked-out or overexpressed or otherwise influenced in view of their expression rate (e.g. by addition of inhibitory molecules, such as small interfering RNAs). Genes may be selectively switched on or off, and the alterations in the contractile functions of the multiring EHT construct are determined.
The invention provides a method for providing force-generating engineered heart tissue rings derived from human cells comprising the steps of:    a) providing undifferentiated human embryonic stem cells;    b) culturing said undifferentiated human embryonic stem cells under conditions which allow for propagating the cells;    c) mixing the cells with a suitable scaffold material in a circular casting mold;    d) culturing the human embryonic stem cells under conditions which allow for differentiation of the cells into cardiac myocytes and formation of a tissue ring structure.
The method can be performed by using the cell lines, media and conditions described in example 6 of the present invention. Particularly, the embryonic stem cells may comprise a selection system and may be subjected to selection before or simultaneously with reconstitution of the EHTs. As explained in more detail below, a suitable scaffold material may be collagen type I. As a matter of course, the human force-generating engineered heart tissue rings resulting from the method may also be subjected to tensile stress as described above with respect to the multiring constructs.
Accordingly, the invention further provides a force-generating engineered heart tissue ring derived from human cells, preferably human stem cells. The tissue ring essentially consists of differentiated human cardiac myocytes and other cell types usually found in heart tissue, such as fibroblasts, smooth muscle cells, endothelial cells, cells of leukocytic origin, like macrophages, etc.) which are embedded by a non-cellular matrix. The presence of cardiac myocytes may be confirmed by detecting one or more of the following proteins: cardiac myosin heavy chain, α-actinin, desmin and/orcardiac troponin I.
According to a preferred embodiment, the force-generating engineered heart tissue ring derived from human cells is used in drug screening assays. For example, the EHT ring derived from human cells can be used to analyze the capability of candidate drugs to interfere with the physiological function of the native cardiac tissue of a mammal. For this, the capability of said candidate drug to enhance or reduce contractile functions of the human EHT is determined. Moreover, the force-generating engineered heart tissue ring derived from human cells may be used in determining the influence of the activity of certain genes on the physiological function of the native cardiac tissue of a mammal (referred to herein as target validation). For this purpose, genes of the cells within the EHT ring (or cells which are used for preparing the same) may be knocked-out or overexpressed or otherwise influenced in view of their expression rate (e.g. by addition of inhibitory molecules, such as small interfering RNAs). Genes may be selectively switched on or off, and the alterations in the contractile functions of the human EHT are determined.
According to a further aspect, the invention provides a device for preparing a multiring engineered heart tissue construct, comprising a plurality of associated suspension means for suspending force-generating engineered heart tissue rings under tensile stress, wherein at least one of each of the associated suspension means is resiliently biased so that the suspended force-generating engineered heart tissue ring is able to contract against the bias force provided by the suspension means, wherein the suspension means are arranged to each other so that each force-generating engineered heart tissue ring has one or more contact points to an adjacent force-generating engineered heart tissue ring when suspended in the device. A preferred embodiment of a device according to the invention is depicted in FIG. 10.
Preferably, two suspension means are associated to each other to form a pair of associated suspension means. It is preferred that the device comprises more than two pairs of associated suspension means so that more than two EHT rings can be suspended in the device. Preferably, the device is design for more than 5 EHT rings, for example 15, 20, 30 or even more.
According to a further embodiment, the associated suspension means of the device are arranged to each other so that the force-generating engineered heart tissue rings can be suspended by stacking to form a central region in which the force-generating engineered heart tissue rings overlap with each other.
According to a particularly preferred embodiment, all associated suspension means are resiliently biased. Preferably, at least one suspension means which is resiliently biased can be adjusted to vary the tensile stress.